Fas peptide mimetics and uses thereof

ABSTRACT

Exocyclic peptide mimetics that disable Fas were developed. A three dimensional model of the Fas receptor-ligand complex was constructed and structurally predicted regions of the receptor that were relevant to binding ligand were used to create constrained peptide mimetics. Exocyclic anti-Fas peptide mimetics were identified that block Fas receptor-ligand interactions, and modulate Fas biological activity both in vitro and in vivo. The mimetics are useful, e.g., for treating Fas-related pathologies.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Priority is claimed under 35 U.S.C. § 119(e) to provisional U.S.patents applications serial No. 60/383,309, filed May 23, 2002, and60/465,943, filed Apr. 28, 2003, both of which are incorporated hereinby reference and in their entireties.

GOVERNMENT SUPPORT

[0002] The work leading to this invention was supported at least in partby grants PO1 CA89480 and RO1 89481 awarded by the National CancerInstitute. The United States government may have certain rights to thisinvention pursuant to the terms of those grants.

FIELD OF THE INVENTION

[0003] The present invention relates to therapeutic agents that act bydisrupting or inhibiting signaling through cell surface receptors. Morespecifically, the invention is directed to peptide mimetics that inhibitsignaling through the Fas receptor, and to methods of using such peptidemimetics to treat Fas-related pathologies.

BACKGROUND OF THE INVENTION

[0004] Fas (CD95/APO-1) and its specific ligand (FasL/CD95L) are membersof the tumor necrosis factor (TNF) receptor and TNF families ofproteins, respectively. (Nagata, S. et al. Science 267, 1449-1456(1995). Interaction between Fas and FasL triggers a cascade ofsubcellular events that results in a definable cell death process inFas-expressing targets. Fas is a 45 kDa type I membrane proteinexpressed constitutively in various tissues, including spleen, lymphnodes, liver, lung, kidney and ovary. (Leithauser, F. et al. Lab Invest69, 415-429 (1993); Watanabe-Fukunaga, R. et al. J Immunol 148,1274-1279 (1992)). FasL is a 40 kDa type II membrane protein, and itsexpression is predominantly restricted to lymphoid organs and perhapscertain immune-privileged tissues. (Suda, T. et al. Cell 75, 1169-1178(1993); Suda, T. et al. J Immunol 154, 3806-3813 (1995)). In humans,FasL can induce cytolysis of Fas-expressing cells, either as amembrane-bound form or as a 17 kDa soluble form, which is releasedthrough metalloproteinase-mediated proteolytic shedding. (Kayagaki, N.et al. J Exp Med 182, 1777-1783 (1995); Mariani, S. M. et al. Eur JImmunol 25, 2303-2307 (1995)).

[0005] The FasL/Fas system has been implicated in the control of theimmune response and inflammation, the response to infection, neoplasia,and death of parenchymal cells in several organs. (Nagata et al. supra;Biancone, L. et al. J Exp Med 186, 147-152 (1997); Krammer, P. H. AdvImmunol 71, 163-210 (1999); Seino, K. et al. J Immunol 161, 4484-4488(1998)). Defects of the FasL/Fas system can limit lymphocyte apoptosisand lead to lymphoproliferation and autoimmunity. A role for FasL-Fas inthe pathogenesis of rheumatoid arthritis, Sjogren's syndrome, multiplesclerosis, viral hepatitis, renal injury, inflammation, aging, graftrejection, HIV infection and a host of other diseases has been proposed.(Famularo, G., et al. Med Hypotheses 53, 50-62 (1999)). Fas mediatedapoptosis is an important component of tissue specific organ damage,such as liver injury which has been shown to be induced through theengagement of the Fas-FasL receptor system. (Kakinuma, C. et al. ToxicolPathol 27, 412-420 (1999); Famularo et al. supra; Martinez, O. M. et al.Int Rev Immunol 18, 527-546 (1999); Kataoka, Y. et al. Immunology 103,310-318 (2001); Chung, C. S. et al. Surgery 130, 339-345 (2001);Doughty, L. et al. Pediatr Res 52, 922-927 (2002)). Consequently, theFasL-Fas pathway represents an important general target for therapeuticintervention.

[0006] Monoclonal anti-FasL antibody and recombinant soluble Fas proteinare well recognized potential candidate antagonists for clinicalstudies. (Hashimoto, H. et al. Arthritis and Rheumatism 41, 657-662(1998); Kanda, Y. et al. Bone Marrow Transplantation 22, 751-754 (1998);Kato, K. et al. British Journal of Haematology 103, 1164-1166(1998);Maggi, C. A. Pharmacological Research 38, 1-34(1998)). Attempts toneutralize FasL with antibodies has been examined in a variety of animalmodels. (Okuda, Y. et al. Biochem Biophys Res Commun 275, 164-168(2000)). While antibodies have a long half life and are highly specificthey also have important limitations: (i) commercial-scale productionmay be either difficult or costly, (ii) conformational stability mayvary with the environment of the body fluids, (iii) antibodies may beexcluded from certain compartments e.g., the brain, due to failure tocross the blood/brain barrier, and (iv) they may lead to the developmentof neutralizing antibodies, etc. (Cho, M. J. et al. Trends Biotechnol14, 153-158 (1996)).

[0007] Many disadvantages of large macromolecules can be overcome bycreating small molecular inhibitors that are targeted to surfacereceptors or their ligands. Peptidomimetics that are constructed toresemble secondary structural features of the targeted protein representan approach to overcome some of the limitations of macromolecules andcan mimic inhibitory features of large molecules such as antibody (Park,B. W. et al. Nat Biotechnol 18, 194-198 (2000)) and soluble receptors.(Takasaki, W. et al. Nat Biotechnol 15, 1266-1270 (1997)). Recentlyseveral peptidomimetics that inhibit ligand-receptor binding and thatmediate potent biological effects have been described. (Park et al.supra; Takasaki, et al. supra). These peptides represent novel smallmolecular tools that can act with potency comparable to or equivalent tothe natural antagonist. (Takasaki et al. supra; Wrighton, N. C. et al.Nat Biotechnol 15, 1261-1265 (1997)).

[0008] Several studies suggest that the presence of FasL in the eye is abarrier to both inflammatory cells (Griffith, T. S., et al., Science,270, 1189-92 (1995); Gao, Y., et al., J Exp Med., 188, 887-96, (1998))and development of new blood vessels (Kaplan, H. J., et al., Nat Med.,5, 292-97 (1999)). The control of inflammation is known to be acomponent of the immune privilege of the eye (Griffith et al., 1995,supra; Griffith, T. S., et al., Immunity, 5, 7-16 (1996); Greil, R., A.,et al., Leukemia & Lymphoma, 31, 477, (1998); Oconnell, J., et al.,Molecular Medicine, 3, 294-300 (1997)). FasL expression in ocular tissueinduces apoptosis in Fas⁺ lymphoid cells that invade the eye in responseto viral infection or corneal grafting (Griffith, T. S., et al., 1995,supra; Stuart, P. M., et al., J Clin Invest, 99, 396-402 (1997); Chen,J. J., et al., Science, 282, 1714-1717 (1998); Mohan, R. R., et al.,Experimental Eye Research, 65, 575-589 (1997)). FasL expression in theretina inhibits growth of blood vessels beneath the retina (Kaplan etal., supra), by inducing apoptosis in vascular endothelial cells thatare known to express the Fas antigen. The loss of FasL expression inthis region may be a predisposing factor in age related maculardegeneration, allowing vessels to localize beneath the retina afterpenetration of Bruch's membrane. This process may lead to retinaldetachment and visual loss.

[0009] FasL is also expressed in the cornea. Corneas that did notexpress functional FasL (gld) showed significantly greaterneovascularization than normal corneas. In addition, engagement of Fason vessels growing in vitro prevents vascular extension. These resultssuggest that FasL regulates neovascularization by engaging Fas ongrowing vessels and inducing apoptosis of the Fas⁺ vascular endothelialcells. Fas/FasL interaction is also required for the antiangiogeniceffects of IL-12 and IL-2 when treating murine renal carcinoma(Wigginton, J. M., et al., J Clin Invest., 108, 51-62 (2001)).

[0010] Corneal neovascularization may be due to a complex interplaybetween several anti-angiogenic factors and Fas. This is evident fromthe fact that while gld and lpr mice are less prone to spontaneousneovascularization, normal development of eye is unaffected. This issimilar to observations made with the immune privilege of the eye, whereFasL works in concert with other inhibitory agents to control the spreadof inflammation (Stuart, P. M., et al., Invest Ophthalmol Vis Sci., 44,93-98 (2003)). The role of FasL seems to be critical when the eye ischallenged or stimulated with an agent that induced inflammation(Griffith et al., 1995, supra; Kaplan et al., supra) or growth factorssuch FGF, HGF etc. (Stuart, et. al., supra).

[0011] Recently, an inhibitor responsible for the avascularity of ocularcompartments was identified in the cornea as pigment epithelium-derivedfactor (PEDF) (Dawson, D. W., et al., Science, 285, 245-48 (1999)). Thisprotein has been shown to have neurotrophic activity (Tombran-Tink, J.,et al., J Neurosci, 15, 4992-5003 (1995); Taniwaki, T., et al., JNeurochem, 68, 26-32 (1997)) but is now known as a potentanti-angiogenic molecule (Gettins, P. G., et al., Biol Chem, 383,1677-82 (2002); Simonovic, M., et al., Proc Natl Acad Sci USA, 98,11131-35 (2001)). It seems to be a constitutive component of ocularcompartments, and neutralization of its activity permits new vesselgrowth into the central cornea (Dawson et al., supra). Apoptosis inendothelial cells is associated with the activity of PEDF (Stellmach,V., et al., Proc Natl Acad Sci USA, 98, 2593-2597 (2001); Volpert, O.V., et al., Nat Med, 8, 349-357 (2002)). It has been shown that PEDFinhibitory function is orchestrated via upregulation of Fas (Volpert, O.V., et al., supra) in the cornea to inhibit spontaneousneovascularization and limit induction of angiogenesis.

[0012] The pattern of Fas L expression in the eye and the resutsobtained when FasL expression is modified in ocular tissue suggest thatCodifying Fas function might be useful for various eye relatedpathologies. Altering Fas function in the eye may be a useful strategy,for example, for inhibiting corneal neovascularization.

[0013] The present inventors have undertaken a structural analysisFas-FasL interactions. The inventors have developed an interaction modelof FasL and Fas created by computer assisted modeling and designedpeptide mimetics based on the deduced secondary structural features ofFas. The understanding of Fas-FasL recognition features at the atomiclevel has allowed the inventors to design mimetics that modulate Fassignaling functions and can thus be used to treat Fas pathologies.

[0014] Accordinly, the present inventors have identified and developedpeptidomimetics that alter Fas function and will therefore havetherapeutic applications against disease states mediated by Fas. Themimetics were effective in inhibiting β-FGF induced cornealneovascularization in vivo, demonstrating that Fas-FasL interactionplays a significant role in regulating extension of new blood vesselsinto the cornea, and indicating that Fas is a therapeutic target for eyerelated pathologies. The in vivo biological activity of the mimetics wasalso validated in a murine model of Fas-dependent Con A inducedhepatitis injury. Accordingly, the mimetics are therefore also useful astherapy for fulminant hepatitis.

[0015] The citation and/or discussion of a reference in this section,and throughout this specification, shall not be construed as anadmission that such reference is prior art to the present invention.

SUMMARY OF THE INVENTION

[0016] In certain embodiments, the invention provides an exocyclicpeptide having an amino acid sequence of a Fas surface domain thatinteracts with FasL.

[0017] In certain embodiments, the invention provides a Fas mimeticrepresented by the formula (I)

[0018] wherein:

[0019] B₁ and B₉ are independently a peptide of 1-6 amino acids, atleast one of which is a hydrophobic amino acid, an aromatic moiety or aheteroaromatic moiety,

[0020] Z₂ is a moiety that is capable of forming a covalent linkage withB₁, X₃ and Z₈,

[0021] Z₈ is a moiety that is capable of forming a covalent linkage withB₉, X₇ and Z₂,

[0022] X₃ is a hydrophilic amino acid or a bond,

[0023] X₄ is an amino acid selected from aspartic acid or glutamic acid,

[0024] X₅ is an amino acid selected from aspartic acid or glutamic acid,

[0025] X₆ is an amino acid selected from the group consisting ofhistidine, lysine, arginine, asparagine or glutamine,

[0026] X₇ is an aromatic moiety or a heteroaromatic moiety,

[0027] “—” is a covalent linkage comprising an amide, substituted amideor an isostere of amide thereof, and

[0028] “═” is a covalent linkage,

[0029] or a pharmaceutically acceptable salt, metabolite or prodrugthereof.

[0030] Preferably, the mimetic of formula (I) is a peptide comprising anamino sequence selected from the group consisting of YCDEGHLCY (SEQ IDNO: 1), YCDEGLCY (SEQ ID NO: 2), YCDEGYFCY (SEQ ID NO: 3), YCDEGEYCY(SEQ ID NO: 4), YCDEHFCY (SEQ ID NO:5), YCDEHGLCY (SEQ ID NO: 6),YCDEHGQCY (SEQ ID NO: 7), YCDEKFCY (SEQ ID NO: 8), and YCDEQFCY (SEQ IDNO: 9), wherein the cysteine residues of said peptide are preferablyjoined by a covalent bond to form a cyclic peptide. More preferably, themimetic of formula (I) is a peptide comprising an amino sequenceselected from the group consisting of YCDEHFCY (SEQ ID NO: 5), YCDEKFCY(SEQ ID NO: 8) and YCDEQFCY (SEQ ID NO: 9) wherein the cysteine residuesof said peptide are preferably joined by a covalent bond to form acyclic peptide.

[0031] In other embodiments, the invention provides mimetics comprisingan amino acid sequence selected from the group consisting of YCNSTVCY(SEQ ID NO: 10), YCDKAEHFCY (SEQ ID NO: 11), YCNTRTQNTCY (SEQ ID NO:12), YCQEKEYCY (SEQ ID NO: 13), and YCQERKEYCY (SEQ ID NO: 14).

[0032] In other embodiments, the invention is directed to a method ofpreventing or treating a FAS-related pathology comprising administeringto a mammal (e.g., a human) suffering from said FAS-related pathology atherapeutically effective amount of the foregoing mimetics.

[0033] The invention also provides pharmaceutical compositionscomprising the aforementioned mimetics.

[0034] In other embodiments, the invention is directed to a method ofinhibiting Fas receptor-Fas ligand interaction comprising exposing Fasor FasL to an effective amount of the aforementioned mimetics to inhibitsaid interaction.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIGS. 1A-B depicts the three dimensional structure of the bindingsite in FasL/Fas complex. (A) Interaction between a FasL dimer and Faspeptide mimetics Kp1, Kp2, Kp3, Kp4 and Kp7. (B) Putative solutionstructure of peptide mimetic Kp7-6.

[0036] FIGS. 2A-B illustrates inhibition of FasL binding to Fas-receptorby exocyclic mimetics in a binding assay.

[0037] FIGS. 3A-B depicts a surface plasmon resonance (biosensor)analysis of mimetic binding to immobilized FasL.

[0038]FIG. 4 depicts inhibition of FasL-induced cytolysis in Jurkatcells by the antagonistic peptides.

[0039]FIG. 5 depicts inhibition of FasL-induced apoptosis in Jurkatcells by the peptide mimetic Kp7-6. The number above the bar in eachpanel indicates the percentage of apoptotic (annexin-V⁺) cells.

[0040] FIGS. 6A-B illustrates results showing protection of mice againstCon A induced liver injury by the antagonistic Fas mimetic peptid Kp7-7as measured by serum activities of (A) alanine aminotransferase and (B)aspartate aminotransferase. *P<0.0001 versus control; **P<0.01 versusCon A-treated mice.

[0041] FIGS. 7A-B shows apopotosis levels measured in (A) N6803 cellsand (B) WS10201 cells treated with anti-Fas IgM antibody and Fas mimeticKp7-6.

DETAILED DESCRIPTION

[0042] The present invention is concerned with mimetics that areantagonists of the Fas receptor (Fas)-Fas ligand (FasL) signaling systemand methods of using such mimetics. The invention is based in part onthe findings that sites on Fas that function in the binding of FasL canbe identified by comparison to the TNF receptor and that peptidemimetics of the sites identified on Fas act to inhibit binding of FasLto Fas and inhibit Fas function, i.e., the mimetics are antagonists ofFasL binding and of Fas signaling and function. The mimetics aretherefore useful in the treatment of Fas-related pathologies.

[0043] Hence, the invention provides exocyclic peptide mimeticscomprising an amino acid sequence of a Fas surface domain that interactswith FasL or derived from an amino acid sequence of a Fas surface domainthat interacts with FasL.

[0044] The invention provides Fas mimetics represented by formula (I)

[0045] wherein:

[0046] B₁ and B₉ are independently a peptide of 1-6 amino acids, atleast one of which is a hydrophobic amino acid, an aromatic moiety or aheteroaromatic moiety,

[0047] Z₂ is a moiety that is capable of forming a covalent linkage withB₁, X₃ and Z₈,

[0048] Z₈ is a moiety that is capable of forming a covalent linkage withB₉, X₇ and Z₂,

[0049] X₃ is a hydrophilic amino acid or a bond,

[0050] X₄ is an amino acid selected from aspartic acid or glutamic acid,

[0051] X₅ is an amino acid selected from aspartic acid or glutamic acid,

[0052] X₆ is an amino acid selected from the group consisting ofhistidine, lysine, arginine, asparagine or glutamine,

[0053] X₇ is an aromatic moiety or a heteroaromatic moiety,

[0054] “—” is a linkage comprising an amide, substituted amide or anisostere of amide thereof, and

[0055] “═” is a covalent linkage,

[0056] or a pharmaceutically acceptable salt, metabolite or prodrugthereof.

[0057] B₁ and B₉ independently are exocyclic portions of mimetics offormula (I) that are comprised of, e.g., exocyclic amino acid residues.Z₂, X₃, X₄, X₅, X₆, X₇ and Z₈ comprise the cyclicized portion ofmimetics of formula (I). Z₂ and Z₈ are further linking moieties,preferably linking amino acids.

[0058] Preferably, Fas mimetics of the invention are conformationallyrestrained peptides. Most preferably, such conformationally restrainedpeptides are cyclicized peptides comprising a cyclicized portion, one ormore exocyclic region, one or more linking moiety and an active region.

[0059] Independent preferred moieties for the formula I are as follows:Z₂ is cysteine; Z₈ is cysteine; X₃ is a bond; X₄ is aspartic acid; X₅ isglutamic acid; X₇ is phenylalanine; and X₇ is an aromatic amino acid.

[0060] The following are also independent preferences: B₁ is tyrosine;B₉ is tyrosine; both B₁ and B₉ are tyrosine; B₁ is —R₁-R₂, where R₁ isan aromatic amino acid linked to Z₂ and R₂ is a peptide of 1-5 aminoacids; B₉ is —R₃-R₄, where R₃ is an aromatic acid linked to Z₈ and R₄ isa peptide of 1-5 amino acids.

[0061] Also preferred is a mimetic of formula (I) comprising an aminosequence selected from the group consisting of YCDEGHLCY (SEQ ID NO: 1),YCDEGLCY (SEQ ID NO: 2), YCDEGYFCY (SEQ ID NO: 3), YCDEGEYCY (SEQ ID NO:4), YCDEHFCY (SEQ ID NO: 5), YCDEHGLCY (SEQ ID NO: 6), YCDEHGQCY (SEQ IDNO: 7), YCDEKFCY (SEQ ID NO: 8) and YCDEQFCY (SEQ ID NO: 9), wherein thecysteine residues of said amino acid sequence are joined by a covalentbond, to form a cyclic peptide. More preferably, a mimetic of formula(I) comprises an amino sequence selected from the group consisting ofYCDEHFCY (SEQ ID NO: 5), YCDEKFCY (SEQ ID NO: 8) and YCDEQFCY (SEQ IDNO: 9), wherein the cysteine residues of said amino acid sequence arejoined by a covalent bond, to form a cyclic peptide.

[0062] Also provided for use in the invention is a mimetic comprising anamino acid sequence selected from the group consisting of YCNSTVCY (SEQID NO: 10), YCDKAEHFCY (SEQ ID NO: 11), YCNTRTQNTCY (SEQ ID NO: 12),YCQEKEYCY (SEQ ID NO: 13), and YCQERKEYCY (SEQ ID NO: 14).

[0063] Preferred mimetics provided in the invention are in the range ofabout 5 to about 100 amino acids in length. Further preferred mimeticsprovided in the invention are in the range of about 5 to about 50 aminoacids in length, or in the range of about 10 to about 50 amino acids inlength. Still further preferred mimetics provided in the invention arein the range of about 5 to about 40 amino acids in length, or in therange of about 10 to about 40 amino acids in length. Still furtherpreferred mimetics provided in the invention are in the range of about 5to about 30 amino acids in length, or in the range of about 10 to about30 amino acids in length. Still further preferred mimetics provided inthe invention are in the range of about 5 to about 20 amino acids inlength, or in the range of about 10 to about 20 amino acids in length.Most preferred mimetics provided in the invention are in the range ofabout 5 to about 10 amino acids in length.

[0064] Definitions:

[0065] Amino acid residues used in the present invention may be recitedby their full name or by reference to either the three letter or singleletter amino acid code (see, e.g., Table 5.1 of Mathews & van Holde,Biochemistry, Second Edition (Benjamin/Cumings Publishing Company, Inc.,New York) at page 131). The mimetics that are encompassed within thescope of the invention are partially defined in terms of amino acidresidues of designated classes. The amino acids may be generallycategorized into three main classes: hydrophilic amino acids,hydrophobic amino acids and cysteine-like amino acids, dependingprimarily on the characteristics of the amino acid side chain. Thesemain classes may be further divided into subclasses. Hydrophilic aminoacids include amino acids having acidic, basic or polar side chains.Hydrophobic amino acids include amino acids having aromatic or apolarside chains. Apolar amino acids may be further subdivided to include,among others, aliphatic amino acids. The definitions of the classes ofamino acids as used herein are as follows:

[0066] The term “hydrophobic amino acid” refers to an amino acid havinga side chain that is uncharged at physiological pH and that is repelledby aqueous solution. Examples of genetically encoded hydrophobic aminoacids include Ile, Leu and Val. Examples of non-genetically encodedhydrophobic amino acids include t-BuA.

[0067] The term “aromatic amino acid” refers to a hydrophobic amino acidhaving a side chain containing at least one ring having a conjugatedpi-electron system (aromatic group). The aromatic group may be furthersubstituted with substituent groups such as alkyl, alkenyl, alkynyl,hydroxyl, sulfanyl, nitro and amino groups, as well as others. Examplesof genetically encoded aromatic amino acids include phenylalanine,tyrosine and tryptophan. Commonly encountered non-genetically encodedaromatic amino acids include phenylglycine, 2-naphthylalanine,β-2-thienylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and4-fluorophenylalanine.

[0068] The term “hydrophilic amino acid” refers to an amino acid havinga side chain that is capable of bonding to solvent molecules in anaqueous solution. Examples of genetically encoded hydrophilic aminoacids include Ser and Lys. Examples of non-encoded hydrophilic aminoacids include Cit and hCys.

[0069] The term “heteroaromatic moiety” refers to an aromatic moietywherein one or more of the ring carbon atoms is replaced with anotheratom such as, for example, nitrogen, oxygen or sulfur. Typicalheteroaromatic moieties include, but are not limited to, pyran,pyrazole, pyridine, pyrrole, pyrazine, pyridazine, pyrimidine,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,selenophene, thiophere, tellurophene, xanthene, and the like.

[0070] Additional examples of amino acids and related compounds arefound in U.S. Pat. No. 6,265,535.

[0071] As used herein, the term “mimetic” refers to compounds whichmimic the /activity of a peptide. Mimetics may themselves be peptides.Mimetics may also be non-peptides and/or may comprise amino acids linkedby non-peptide bonds, e.g., without limitation, psi bonds (see, e.g.,Benkirane, N., et al. J. Biol. Chem., 271:33218-33224 (1996)). U.S. Pat.No. 5,637,677 and its parent applications contain detailed guidance onthe production of mimetics. Preferred mimetics are “conformationallyconstrained” peptides. Also preferred are cyclic mimetics. Furtherpreferred are cyclic mimetics comprising at least one exocyclic domain,a linking moiety (e.g., a linking amino acid) and an active region.

[0072] As used herein, the terms “constrained peptides” and“conformationally constrained peptides” are used interchangeably and aremeant to refer to peptides which, e.g., through intramolecular bonds,are conformationally stable and remain in a restricted conformation.

[0073] As used herein, the term “exocyclic amino acid residue” is meantto refer to an amino acid residue that is linked directly or indirectlyto a cyclicized peptide but which is not within the portion of thepeptide that makes up the circularized structure.

[0074] As used herein, the term “exocyclic portion” is meant to refer toan amino acid sequence having one or more amino acid residues which arelinked to cyclicized peptide but which are not within the portion of thepeptide that makes up the circularized structure.

[0075] As used herein, the term “linking moiety” is meant to refer to amolecular component or functional group which is capable of formingbonds with three amino acids.

[0076] As used herein, the term “linking amino acid residue” is meant torefer to an amino acid residue that is a linking moiety.

[0077] As used herein, the term “active region” is meant to refer to theamino acid sequence of the portion of a mimetic that directly interactswith a receptor or receptor ligand, wherein the interaction ischaracterized by an affinity between the active region of the mimeticand the receptor or receptor ligand.

[0078] The term “Fas mimetic” refers to a peptide mimetic that isderived from a Fas peptide; i.e., a Fas mimetic of the invention is acompound that mimics a Fas peptide (a peptide derived from Fas).Preferred Fas mimetics are mimetics of a peptide derived from and/orcorresponding to a Fas surface domain. Further preferred are Fasmimetics that are mimetics of a peptide derived from and/orcorresponding to a Fas surface loop domain. Exemplary Fas peptides areprovided, infra, in Table 1. Particularly preferred Fas mimetics of theinvention are mimetics of Fas Kp7 surface domain peptides.

[0079] Fas mimetics are preferably derived from human or mouse Fas. Morepreferably, Fas mimetics are derived from human Fas, e.g., withoutlimitation, human Fas. Most prefereably, mimetics are derived from humanFas with the mature amino acid sequence:RLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHH (SEQ ID NO: 15)DGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITS DSENSNFRNEIQSLV

[0080] Preferred Fas mimetics of the invention are capable ofinteracting with (e.g., binding to) either Fas, FasL or both Fas andFasL. Fas mimetics may therefore modulate Fas activity (in particular,Fas mediated signaling), for example as antagonists, agonists, orinverse agonists. For example, in certain embodiments a Fas mimetic ofthe invention may inhibit binding of FasL to Fas, and may therebyinhibit Fas activity. In other embodiments (that are preferablypracticed either in the absence of or at very low concentrations ofFasL) a Fas mimetic of the invention may bind to and activate Fas.

[0081] As used herein, the term “surface domain” refers to a domain of aprotein comprising one or more solvent accessible amino acid. A surfacedomain make include, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 solvent accessible amino acids. A surfacedomain may also include greater than 20 solvent accessible amino acids.Preferably, each amino acid in a surface domain is a solvent accessibleamino acid. Solvent accessible amino acids and hence surface domains maybe identified using methods well known in the art (see, e.g., Jones etal., J. Mol. Biol., 272:121-132 (1997) and Samanta, U. et al., Prot.Eng., 15:659-667 (2002)).

[0082] Solvent accessibility of an amino acid is expressed as a valuefrom 0.0 (buried) to 1.0 completely accessible. Preferably, a surfacedomain comprises at least one amino acid with a solvent accessibility ofat least about 0.3. More preferably, a surface domain comprises at leastone amino acid with a solvent accessibility of at least about 0.4, 0.5,0.6 or 0.7. More preferably, a surface domain comprises at least oneamino acid with a solvent accessibility of at least about 0.8, 0.9 or0.95. Further preferred is where a surface domain comprises about 2, 3,4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of atleast 0.3, or comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids with a solvent accessibility of at least about 0.3. Furtherpreferred is where a surface domain comprises about 2, 3, 4, 5, 6, 7, 8,9, or 10 amino acids with a solvent accessibility of at least about 0.4,0.5, 0.6, or 0.7, or comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids with a solvent accessibility of at least about 0.4, 0.5,0.6, or 0.7. Further preferred is where a surface domain comprises about2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibilityof at least about 0.8, 0.9, or 0.95, or comprises at least about 2, 3,4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of atleast about 0.8, 0.9, or 0.95.

[0083] Also preferred is where a surface domain consists of about 2, 3,4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of atleast about 0.3, or consists of at least about 2, 3, 4, 5, 6, 7, 8, 9,or 10 amino acids with a solvent accessibility of at least about 0.3.Further preferred is where a surface domain consists of about 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of atleast about 0.4, 0.5, 0.6, or 0.7, or consists of at least about 2, 3,4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of atleast about 0.4, 0.5, 0.6, or 0.7. Further preferred is where a surfacedomain consists of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids witha solvent accessibility of at least about 0.8, 0.9, or 0.95, or consistsof at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with asolvent accessibility of at least about 0.8, 0.9, or 0.95.

[0084] As used herein, the term “treatment” refers to administering anactive agent to lessen the severity or the likelihood of there-occurrence of a pre-existing condition. Hence, “treatment”encompasses, for example and without limitation, ameliorating at leastone symptom associated with a condition or reducing the rate ofoccurrence or re-occurrence of a condition.

[0085] As used herein, the term “preventing” refers to the lessening ofthe likelihood of the occurrence of a condition.

[0086] As used herein, a “subject in need of treatment of” or a “subjectsuffering from” a condition is a mammal (e.g., human) that manifests atleast one symptom of a condition or that is at risk of developing orre-developing the particular condition to be treated.

[0087] As used herein, a “therapeutically effective amount” of an agentis an amount sufficient to ameliorate at least one symptom associatedwith a pathological, abnormal or otherwise undesirable condition, anamount sufficient to prevent or lessen the probability that such acondition will occur or re-occur, or an amount sufficient to delayworsening of such a condition.

[0088] As used herein, a “Fas-related pathology” is a pathologicalcondition that can be treated by increasing or decreasing activity(i.e., signaling) of Fas. A “Fas-related pathology” is preferablytreated with an antagonist of Fas activity. More preferably, saidantagonist binds to one or both of Fas or FasL to lower Fas activity.Alternatively, in certain embodiments, a “Fas-related pathology” may betreated with an agonist of Fas activity. Examples of “Fas-relatedpathologies” include, without limitation, pathologies related tolymphocyte apoptosis leading to abnormal lymphoproliferation andautoimmunity. Additional examples include, without limitation, aging andcell death (e.g., in the skin and other organs), visual loss, rheumatoidarthritis, Sjogren's syndrome, multiple, viral hepatitis, renal injury,HIV infection, and angiogenesis. See also Famularo et al. supra. Theinhibition of FasL and Fas antigen interactions has also been used toprevent transplantation rejections. Mimetics of the invention whichinhibit the binding of Fas with FasL may be used to augment immuneresponses in certain settings.

[0089] In a preferred embodiment, memetics of the invention are used totreat visual loss or other ocular disorder either with or withoutcoincident neovascularization. In a preferred embodiments, the mimeticsof the invention are used to treat visual loss or other ocular disorderwith coincident neovascularization. Examples of ocular disorder include,without limitation, macular degeneration, e.g, age-related maculardegeneration.

[0090] A “metabolite” of a compound disclosed herein is a derivative ofa compound which is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound which is formed when the compound is metabolized. The term“metabolized” refers to the sum of the processes by which a particularsubstance is changed in the living body. In brief, all compounds presentin the body are manipulated by enzymes within the body in order toderive energy and/or to remove them from the body. Specific enzymesproduce specific structural alterations to the compound. For example,cytochrome P450 catalyses a variety of oxidative and reductive reactionswhile uridine diphosphate glucuronyltransferases catalyze the transferof an activated glucuronic-acid molecule to aromatic alcohols, aliphaticalcohols, carboxylic acids, amines and free sulfhydryl groups. Furtherinformation on metabolism may be obtained from The Pharmacological Basisof Therapeutics, 9th Edition, McGraw-Hill (1996), pages 11-17.

[0091] Metabolites of the compounds disclosed herein can be identifiedeither by administration of compounds to a host and analysis of tissuesamples from the host, or by incubation of compounds with hepatic cellsin vitro and analysis of the resulting compounds. Both methods are wellknown in the art.

[0092] A “prodrug” of a compound disclosed herein is an inactivesubstance that converts into an active form of the disclosed compoundsin vivo when administered to a mammal.

[0093] The compounds of the present invention are related to mimetics ofFas as disclosed above, including all enantiomers, diastereomers,crystalline forms, hydrates, solvates or pharmaceutically acceptablesalts thereof, as well as active metabolites of these mimetics havingthe same type of activity.

[0094] An antagonist of Fas is a substance which diminishes or abolishesthe effect of a ligand (agonist) which typically activates Fas receptor,e.g., FasL. The antagonist may be, for example, a chemical antagonist, apharmacokinetic antagonist, an antagonist by receptor block, anon-competitive antagonist or a physiological antagonist. In a preferredembodiment, the antagonist is a chemical antagonist or an antagonist byreceptor block. The antagonist is preferably a Fas mimetic. Furtherpreferred, the antagonist is a mimetic of Fas peptide Kp1, Kp2, Kp3,Kp4, or Kp7.

[0095] A chemical antagonist is a substance wherein the antagonist bindsthe ligand in solution so the effect of the ligand is lost. Apharmacokinetic antagonist is one which effectively reduces theconcentration of the ligand at its site of action, for example, byincreasing the rate of metabolic degradation of the active ligand.Antagonism by receptor-block involves two important mechanisms:reversible competitive antagonism and irreversible, or non-equilibriumcompetitive antagonism. Reversible competitive antagonism occurs whenthe rate of dissociation of the antagonist molecules is sufficientlyhigh such that, on addition of the ligand, displacement of chemicalantagonist molecules from the receptors effectively occurs. Of coursethe ligand cannot evict a bound antagonist molecule, or vice versa.Irreversible or non-equilibrium competitive antagonism occurs when theantagonist dissociates very slowly, or not at all, from the receptorwith the result that no change in the antagonist occupancy takes placewhen the ligand is applied. Thus, the antagonism is insurmountable.Non-competitive antagonism describes the situation where the antagonistexerts its blocking action at some point in the signal transductionpathway leading to the production of a response by the ligand.

[0096] Mimetics of the invention are preferably antagonists of Fas. Morepreferably, mimetics used in the invention are selective antagonists ofFas. A selective antagonist of Fas is one which antagonizes Fas, butantagonizes other receptors of the TNF family of receptors only weaklyor substantially not at all. Most preferred mimetic antagonists arethose which selectively antagonize Fas receptor at low concentration,for example, those that cause a level of antagonism of 50% or greater ata concentration of 1000 mM or less. Selective Fas mimetic antagonistscan thus typically exhibit at least a 10-fold, preferably a 100-fold andmost preferably a 1000-fold greater activity for Fas than at other TNFreceptors.

[0097] Preferred Embodiments:

[0098] Fas mimetics of the invention preferably have one or more of thefollowing properties:

[0099] (1) Significant Inhibition of FasL Binding to Fas:

[0100] Mimetics preferably exhibit antagonist potency (measured as IC₅₀)between 1 nM and 500 mM. Without limiting the present disclosure, asdescribed in more detail below, potency may be measured by determiningthe antagonist activity of mimetics in vivo or in vitro, including cellextracts or fractions of extracts. Inhibitory potency may also bedetermined using, as non-limiting examples, native or recombinant Fas,and/or soluble Fas. Fas binding may be determined using methods that arewell known to those skilled in the art, such as ELISA and proliferationby MTT (Hansen et al., J. Immunol. Methods 199, 203-210 (1989))

[0101] (2) Selectivity:

[0102] Preferred mimetics exhibit at least about 10-fold greaterantagonist potency for Fas, compared to other TNF receptors. Morepreferred are compounds that exhibit about 100-fold greater antagonistfor Fas, compared to other TNF receptors. Most preferred are compoundsthat exhibit about 1000-fold greater antagonist for Fas, compared toother TNF receptors.

[0103] (3) Binding to FasL:

[0104] Mimetics preferably bind to FasL and inhibit the interaction ofFasL and Fas. The binding affinity of mimetics to FasL can be describedby different parameters, e.g., k_(on) k_(off) and K_(D). In preferredembodiments, the mimetics have affinities for FasL represented by valuesof k_(on) greater than 10 M⁻¹ s⁻¹, k_(off) of less than 10⁻³ s⁻¹ orK_(D) of less than 10⁻⁴ M. More preferably, mimetics have affinities forFasL represented by values of k_(on) greater than 10² M⁻¹ s⁻¹, k_(off)of less than 10⁻⁴ s⁻¹ or K_(D) of less than 10⁻⁵ M. Most preferably,mimetics have affinities for FasL represented by values of k_(on)greater than 10³ M⁻¹ s⁻¹, k_(off) of less than 10⁻⁵ s⁻¹ or K_(D) of lessthan 10⁻⁶ M.

[0105] Accordingly, mimetics having one or more of these properties arecandidates for use in treatment of Fas-related pathologies in mammalsand especially in humans.

[0106] Mimetics with one or more of the above properties can further betested for biological activity using assays that measure Fas function invitro or in vivo.

[0107] Measurement of mimetic biological activity can be determined invitro, e.g., by measuring inhibition of Fas-L induced ctyotoxicity or byinhibition of Fas-L induced apoptosis in cell culture, as describedbelow in Examples 4 and 5, respectively. Alternatively, mimeticbiological activity may also be measured by directly or indirectlymeasuring signaling aspects of Fas activity, such as caspase activity,NF-κB activation, and/or other downstream mediators of Fas signaling.

[0108] A useful animal model for measurement of Fas activity in vivo isthe Con A-induced hepatitis model described in Example 6. This assay isa murine model of human autoimmune hepatitis. (Tiegs, G. et al. J ClinInvest 90, 196-203 (1992)). T cell activation plays a crucial role inthe process of Con A-induced hepatitis, because severe combinedimmunodeficiency disorder (SCID) mice and athymic nude mice, which lackmature T cells, are resistant to the damage induced by Con A. (Tiegs etal. supra; Mizuhara, H. et al. J Exp Med 179, 1529-1537 (1994). Thehepatic injury seems to be induced by several different mechanisms, suchas those involving Fas-FasL (Seino, K. et al. Gastroenterology 113,1315-1322 (1997); Tagawa, Y. et al. Eur J Immunol 28, 4105-4113 (1998);Tagawa, Y. et al. J Immunol 159, 1418-1428 (1997)), theperforin-granzyme system (Watanabe, Y. et al. Hepatology 24, 702-710(1996)), IFN-γ (Tagawa et al. supra; Kusters, S. et al. Gastroenterology111, 462-471 (1996)) and TNF-α-mediated cytotoxicity (Mizuhara et al.supra; Toyabe, S. et al. J Immunol 159, 1537-1542 (1997); Gantner, F. etal. Exp Cell Res 229, 137-146 (1996); Gantner, F. et al. Hepatology 21,190-198 (1995); Kusters, S. et al. Eur J Immunol 27, 2870-2905 (1997);Ksontini, R. et al. J Immunol 160, 4082-4089 (1998)). (Mizuhara, H. etal. J Exp Med 179, 1529-1537 (1994). Hepatic damage is primarilydependent upon the Fas-FasL system, because FasL defective gld/gld miceor Fas defective lpr/lpr mice are resistant to liver injury induced byCon A treatment. (Tagawa et al. supra; Tagawa et al. supra).

[0109] Design of Mimetics:

[0110] According to preferred embodiments a mimetic is designed based ona known region of FAS. In preferred embodiments, the mimetic mimics anextracellular domain of an Fas, more preferably a cystine knot region ofFas. Most preferably, the mimetic is based on the Fas Kp7 domain.

[0111] Identification of Fas peptides on which to base mimetics can beperformed using computer modeling and structural analysis. For instance,Example 1, infra, describes one embodiment in which an interaction modelof FasL and Fas was created by computer assisted modeling, and used todesign peptide mimetics based on deduced secondary structural featuresof Fas. In such an embodiment, a skilled user may define optimalinteractions of Fas surfaces that are consistent with either availablebiological data or structural surface complementarity. Peptide mimeticscan then be designed and synthesized for all secondary structuresinvolved in such interactions, typically corresponding to about fivemimetic compounds. Each compound may be modified as described, e.g., inU.S. Pat. Nos. 6,100,377 and 6,265,535. The mimetic compounds thusobtained can then be tested, e.g., using methods described in thisapplication, for their activity as Fas agonists or antagonists.

[0112] Reducing a macromolecule to a small molecule with similarfunction is a general chemical problem. There have been some attempts todesign mini-proteins by transplanting functional units onto suitablescaffolds (Vita et al., Biopolymers 47, 93-100 (1998)) and minimizingantibodies to single chain antibodies (Magliani et al., NatureBiotechnol. 15, 155-158 (1997)).

[0113] It is now established that conformation constrained peptides aremore bioactive than unconstrained ones. In recent years, the chemistryof peptide cyclization by solid phase synthesis and other methods hasimproved dramatically (Burgess et al. (1996) in Solid phase syntheses ofpeptidomimetics (American Chemical Society, Washington D.C.) pp.ORGN-157; Goodman & Shao, Pure Appl. Chem. 68, 1303-1308 (1996);Hanessian et al., Tetrahedron 53, 12789-12854 (1997); Koskinen &Hassila, Acta Chem. Scand. 50, 323-327 (1996); Kuhn et al., Tetrahedron53, 12497-12504 (1997); Waid et al., Tetrahedron Lett. 37, 4091-4094(1996); Zuckermann, Curr. Opin. Struct. Biol. 3, 580-584 (1993))providing more opportunity to develop peptides into peptidomimetics.

[0114] General principles to create cyclic peptide mimetics that havebeen adopted and in some cases (such as eptifitimide (Integrilin) aglycoprotein 11b 111a inhibitor (COR Therapeutics)) have becomeclinically available. Aromatic residues placed at the termini ofcyclically constrained small peptides increase the activity of mimetics(Takasaki et al., Nat. Biotechnol. 15, 1266-1270 (1997); Zhang et al.,Nature Biotechnol. 14, 472-475 (1996)). Employing such modifications hasallowed creation of high affinity mimetics of antibody mimics based onCDRs (Park et al., Nature Biotechnol. 18, 194-198 (2000)), CD4 (Zhang etal., Nature Biotechnol. 15, 150-154 (1997)), IL4 receptor loop mimetics,anti-CD3 antibody mimics, and TNF cystine knot mimetic that affect TNFαbinding to its receptor (Takasaki et al., Nature Biotechnol. 15,1266-1270 (1997)).

[0115] Chemical Synthesis:

[0116] The mimetics of the invention may be prepared using virtually anyart-known technique for the preparation of peptides and peptideanalogues. For example, the peptides may be prepared in linear ornon-cyclized form using conventional solution or solid phase peptidesyntheses and cyclized using standard chemistries. Preferably, thechemistry used to cyclize the peptide will be sufficiently mild so as toavoid substantially degrading the peptide. Suitable procedures forsynthesizing the peptides described herein as well as suitablechemistries for cyclizing the peptides are well known in the art.

[0117] Formation of disulfide linkages, if desired, is generallyconducted in the presence of mild oxidizing agents. Chemical, enzymaticor photolytic oxidation agents may be used. Various methods are known inthe art, including those described, for example, by Tam, J. P. et al.,1979, Synthesis 955-957; Stewart et al., 1984, Solid Phase PeptideSynthesis, 2d Ed., Pierce Chemical Company Rockford, Ill.; Ahmed et al.,1975, J. Biol. Chem. 250:8477-8482; and Pennington et al., 1991 Peptides1990 164-166, Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands. Anadditional alternative is described by Kamber et al., 1980, Helv ChimActa 63:899-915. A method conducted on solid supports is described byAlbericio, 1985, Int. J. Peptide Protein Res. 26:92-97. Any of thesemethods may be used to form disulfide linkages in the peptides of theinvention.

[0118] Recombinant Synthesis:

[0119] If the peptide is composed entirely of gene-encoded amino acids,or a portion of it is so composed, the peptide or the relevant portionmay also be synthesized using conventional recombinant geneticengineering techniques. The isolated peptides, or segments thereof, arethen condensed, and oxidized, as previously described, to yield a cyclicpeptide.

[0120] For recombinant production, a polynucleotide sequence encoding alinear form of the peptide is inserted into an appropriate expressionvehicle, i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence, or in thecase of an RNA viral vector, the necessary elements for replication andtranslation. The expression vehicle is then transfected into a suitabletarget cell which will express the linear form of the cyclic peptide.Depending on the expression system used, the expressed peptide is thenisolated by procedures well-established in the art. Methods forrecombinant protein and peptide production are well known in the art(see, e.g., Maniatis et al., 1989, Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel et al., 1989,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, N.Y.).

[0121] A variety of host-expression vector systems may be utilized toexpress the peptides described herein. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems.

[0122] The expression elements of the expression systems vary in theirstrength and specificities. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationelements, including constitutive and inducible promoters, may be used inthe expression vector. For example, when cloning in bacterial systems,inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac(ptrp-lac hybrid promoter) and the like may be used; when cloning ininsect cell systems, promoters such as the baculovirus polyhedronpromoter may be used; when cloning in plant cell systems, promotersderived from the genome of plant cells (e.g., heat shock promoters; thepromoter for the small subunit of RUBISCO; the promoter for thechlorophyll a/b binding protein) or from plant viruses (e.g., the 35SRNA promoter of CaMV; the coat protein promoter of TMV) may be used;when cloning in mammalian cell systems, promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5 K promoter) may be used; when generating cell lines thatcontain multiple copies of expression product, SV40-, BPV- and EBV-basedvectors may be used with an appropriate selectable marker.

[0123] In cases where plant expression vectors are used, the expressionof sequences encoding the peptides of the invention may be driven by anyof a number of promoters. For example, viral promoters such as the 35SRNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature310:511-514), or the coat protein promoter of TMV (Takamatsu et al.,1987, EMBO J. 6:307-311) may be used; alternatively, plant promoterssuch as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.3:1671-1680; Broglie et al., 1984, Science 224:838-843) or heat shockpromoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986,Mol. Cell. Biol. 6:559-565) may be used. These constructs can beintroduced into plant cells using Ti plasmids, Ri plasmids, plant virusvectors, direct DNA transformation, microinjection, electroporation,etc. For reviews of such techniques see, e.g., Weissbach & Weissbach,1988, Methods for Plant Molecular Biology, Academic Press, NY, SectionVIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch. 7-9.

[0124] In one insect expression system that may be used to produce thepeptides of the invention, Autographa californica nuclear polyhidrosisvirus (AcNPV) is used as a vector to express the foreign genes. Thevirus grows in Spodoptera frugiperda cells. A coding sequence may becloned into non-essential regions (for example the polyhedron gene) ofthe virus and placed under control of an AcNPV promoter (for example,the polyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (e.g., see Smith et al., 1983, J. Virol. 46:584;Smith, U.S. Pat. No. 4,215,051). Further examples of this expressionsystem may be found in Current Protocols in Molecular Biology, Vol. 2,Ausubel et al., eds., Greene Publish. Assoc. & Wiley Interscience.

[0125] In mammalian host cells, a number of viral based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. (e.g., See Logan et al., 1984, Proc. Natl.Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promotermay be used, (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci.USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicaliet al., 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).

[0126] Other expression systems for producing linear or non-cyclizedforms of the cyclic peptides of the invention will be apparent to thosehaving skill in the art.

[0127] Purification of the Peptides and Peptide Analogues:

[0128] The peptides and peptide analogues of the invention can bepurified by art-known techniques such as high performance liquidchromatography, ion exchange chromatography, gel electrophoresis,affinity chromatography and the like. The actual conditions used topurify a particular peptide or analogue will depend, in part, on factorssuch as net charge, hydrophobicity, hydrophilicity, etc., and will beapparent to those having skill in the art.

[0129] For affinity chromatography purification, any antibody whichspecifically binds the peptides or peptide analogues may be used. Forthe production of antibodies, various host animals, including but notlimited to rabbits, mice, rats, etc., may be immunized by injection witha linear or cyclic peptide. The peptide may be attached to a suitablecarrier, such as BSA, by means of a side chain functional group orlinkers attached to a side chain functional group. Various adjuvants maybe used to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacilli Calmette-Guerin) andCorynebacterium parvum.

[0130] Monoclonal antibodies to a peptide may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Koehler and Milstein,1975, Nature 256:495-497, the human B-cell hybridoma technique, Kosboret al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl.Acad. Sci. USA 80:2026-2030 and the EBV-hybridoma technique (Cole etal., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96 (1985)). In addition, techniques developed for the productionof “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.USA 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda etal., 1985, Nature 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to producecyclic peptide-specific single chain antibodies.

[0131] Antibody fragments which contain deletions of specific bindingsites may be generated by known techniques. For example, such fragmentsinclude but are not limited to F(ab′).sub.2 fragments, which can beproduced by pepsin digestion of the antibody molecule and Fab fragments,which can be generated by reducing the disulfide bridges of theF(ab′).sub.2 fragments. Alternatively, Fab expression libraries may beconstructed (Huse et al., 1989, Science 246:1275-1281) to allow rapidand easy identification of monoclonal Fab fragments with the desiredspecificity for the cyclic peptide of interest.

[0132] The antibody or antibody fragment specific for the desired cyclicpeptide can be attached, for example, to agarose, and theantibody-agarose complex is used in immunochromatography to purifycyclic peptides of the invention. See, Scopes, 1984, ProteinPurification: Principles and Practice, Springer-Verlag N.Y., Inc., NY,Livingstone, 1974, Methods Enzymology: Immunoaffinity Chromatography ofProteins 34:723-731.

[0133] Formulation and Routes of Administration:

[0134] The compounds of the invention, may be administered to a subjectper se or in the form of a pharmaceutical composition. Pharmaceuticalcompositions comprising the compounds of the invention may bemanufactured by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. Pharmaceutical compositions may be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the active peptides or peptide analogues into preparationswhich can be used pharmaceutically. Proper formulation is dependent uponthe route of administration chosen.

[0135] For topical administration the compounds of the invention may beformulated as solutions, gels, ointments, creams, suspensions, etc., asare well-known in the art.

[0136] Systemic formulations include those designed for administrationby injection, e.g., subcutaneous, intravenous, intramuscular,intrathecal or intraperitoneal injection, as well as those designed fortransdermal, transmucosal, oral or pulmonary administration.

[0137] For injection, the compounds of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hanks's solution, Ringer's solution, or physiological salinebuffer. The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

[0138] Alternatively, the compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0139] For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

[0140] For oral administration, the compounds can be readily formulatedby combining the active peptides or peptide analogues withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a patient to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, such aslactose, sucrose, mannitol and sorbitol; cellulose preparations such asmaize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulatingagents; and binding agents. If desired, disintegrating agents may beadded, such as the cross-linked polyvinylpyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

[0141] If desired, solid dosage forms may be sugar-coated orenteric-coated using standard techniques.

[0142] For oral liquid preparations such as, for example, suspensions,elixirs and solutions, suitable carriers, excipients or diluents includewater, glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added.

[0143] For buccal administration, the compounds may take the form oftablets, lozenges, etc., formulated in conventional manner.

[0144] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray from pressurized packs or a nebulizer, with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0145] The compounds may also be formulated in rectal or vaginalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

[0146] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0147] Alternatively, other pharmaceutical delivery systems may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles that may be used to deliver peptides and peptide analogues ofthe invention. Certain organic solvents such as dimethylsulfoxide alsomay be employed, although usually at the cost of greater toxicity.Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid polymers containing thetherapeutic agent. Various of sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

[0148] As the mimetics of the invention may contain charged side chainsor termini, they may be included in any of the above-describedformulations as the free acids or bases or as pharmaceuticallyacceptable salts. Pharmaceutically acceptable salts are those saltswhich substantially retain the antimicrobial activity of the free basesand which are prepared by reaction with inorganic acids. Pharmaceuticalsalts tend to be more soluble in aqueous and other protic solvents thanare the corresponding free base forms.

[0149] Effective Dosage:

[0150] The compounds of the invention will generally be used in anamount effective to achieve the intended purpose. For use to treat orprevent FAS-related pathologies, the compounds of the invention, orpharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. By therapeutically effective amount ismeant an amount effective ameliorate or prevent the symptoms, or prolongthe survival of, the patient being treated. Determination of atherapeutically effective amount is well within the capabilities ofthose skilled in the art.

[0151] For systemic administration, a therapeutically effective dose canbe estimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture (i.e., theconcentration of test compound that inhibits 50% of Fas:Fas-L bindinginteractions). Such information can be used to more accurately determineuseful doses in humans.

[0152] Initial dosages can also be estimated from in vivo data, e.g.,animal models, using techniques that are well known in the art. Onehaving ordinary skill in the art could readily optimize administrationto humans based on animal data.

[0153] Dosage amount and interval may be adjusted individually toprovide plasma levels of the compounds which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.01 to about 25 mg/kg/day, preferably fromabout 0.1 to about 10 mg/kg/day and more preferably from about 0.5 toabout 5 mg/kg/day. Also preferred are total daily dosages from about 25to about 1000 mg per day, preferably from about 100 to about 750 mg perday and more preferably from about 250-500 mg per day. Therapeuticallyeffective serum levels may be achieved by administering multiple doseseach day.

[0154] In cases of local administration or selective uptake, theeffective local concentration of the compounds may not be related toplasma concentration. One having skill in the art will be able tooptimize therapeutically effective local dosages without undueexperimentation.

[0155] The amount of compound administered will, of course, be dependenton the subject being treated, on the subject's weight, the severity ofthe affliction, the manner of administration and the judgment of theprescribing health professional.

[0156] The therapy may be repeated intermittently while symptoms aredetectable or even when they are not detectable. The therapy may beprovided alone or in combination with other drugs. In the case ofFas-related pathologies, the drugs that may be used in combination withthe mimetics of the invention include, but are not limited to,anti-inflammatories, steroids and antimetabolites (for example,methotrexate).

[0157] Drugs used “in combination” are administered as part of a commonmethod of treating a given pathology. Drugs used in combination may beadministered in single or separate dosage forms. Separate dosage formsmay be administered at the same or different times.

[0158] Toxicity:

[0159] Preferably, a therapeutically effective dose of the compoundsdescribed herein will provide therapeutic benefit without causingsubstantial toxicity.

[0160] Toxicity of the compounds described herein can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) or the LD₁₀₀ (the dose lethal to 100% of the population).The dose ratio between toxic and therapeutic effect is the therapeuticindex. Compounds which exhibit high therapeutic indices are preferred.The data obtained from these cell culture assays and animal studies canbe used in formulating a dosage range that is not toxic for use inhuman. The dosage of the compounds described herein lies preferablywithin a range of circulating concentrations that include the effectivedose with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basisof Therapeutics, Ch.1, p.1).

EXAMPLES

[0161] The present invention is also described by means of the followingexamples. However, the use of these or other examples anywhere in thespecification is illustrative only and in no way limits the scope andmeaning of the invention or of any exemplified term. Likewise, theinvention is not limited to any particular preferred embodimentsdescribed herein. Indeed, many modifications and variations of theinvention may be apparent to those skilled in the art upon reading thisspecification and can be made without departing from its spirit andscope. The invention is therefore to be limited only by the terms of theappended claims along with the full scope of equivalents to which theclaims are entitled.

Example 1 Molecular Model of a Fas Receptor Complex

[0162] Fas, a member of TNF superfamily, shares significant structuralhomology with the TNF receptor. The structure of the TNF receptorcontains distinct “cystine-knot” repeating domains. (Naismith, J. H. etal. Structure 4, 1251-1262 (1996)). Loop structures in the first threedomains as well as β-turns in proteins are considered to mediate rolesin molecular recognition and binding. (Leszczynski, J. F. et al. Science234, 849-855 (1986)). To develop a cystine-knot peptide mimetic, sitesof protein-protein interaction that might be disrupted or influenced bysmall molecules were identified.

[0163] A molecular model of the Fas and FasL complex (FIG. 1a) wasdeveloped using the crystal structure of TNF receptor complex in ourmethodology as well as other already published models.(Watanabe-Fukunaga et al. supra; Naismith, J. H. et al. J Biol Chem 270,13303-13307 (1995); Banner, D. W. et al. Cell 73, 431-445 (1993);Bajorath, J. J Comput Aided Mol Des 13, 409-418 (1999)). The overallfeatures of the receptor-ligand interaction were noted to be verysimilar to that of TNF receptor ligand complex. See, U.S. Pat. No.6,265,535. However, the ectodomain of the Fas receptor appears to berotated about 10° compared to the transmembrane domain. Fas-FasL contactsites predicted by the molecular model are consistent with the mutationanalysis data. (Beltinger, C. et al. British Journal of Haematology 102,722-728 (1998)).

[0164] The Fas-FasL structural model suggested five surfaces where FasLcan bind to Fas, designated Kp1, Kp2, Kp3, Kp4 and Kp7 (FIG. 1b),compared to such three sites identified in the TNF receptor (Takasaki etal. supra). The amino acids in the loops Kp1, 2, 3, 4 and 7 adopt welldefined conformations (i.e., adopt statistically allowed conformations)as judged by Ramachandran plots (Ramachandran, G. N. et al. Biopolymers6, 1255-1262 (1968); Ramachandran, G. N. et al. Adv Protein Chem 23,283-438 (1968)) and profile analyses. (Zhang, K. Y. et al. Protein Sci3, 687-695 (1994)).

[0165] Peptide analogs were designed from the various loop structures.Each mimetic was optimized for its ability to mimic the bindingconformation of the loop and for its ring size which we have determinedto be critical to reduce the inherent flexibility of mimetics. Specificfeatures optimized included conformational mimicry between the loopstructure and the mimetic, hydropathic value of the mimetics,dissociation rate (as measured from surface plasmon resonance; see.,Example 3, infra), stability and solubility. A set of mimetics that wereselected for biological assay is shown in Table 1. TABLE 1 ExocyclicMimetics Derived from Fas Fas Receptor Peptide Exocyclic MimeticDesignation Sequence* Designation Sequence Kp1 119 CNSTVC 124 (SEQ IDNO: 16) Kp1-1 YCNSTVCY (SEQ ID NO: 10) Kp2 77 DKAHFSSKC 85 (SEQ ID NO:17) Kp2-2 YCDKAEHFCY (SEQ ID NO: 11) Kp3 103 CTRTQ 107 (SEQ ID NO: 18)Kp3-2 YCNTRTQNTCY (SEQ ID NO: 12) Kp4 69 CQEGKEY 75 (SEQ ID NO: 19)Kp4-2 YCQEKEYCY (SEQ ID NO: 13) Kp4-3 YCQERKEYCY (SEQ ID NO: 14) Kp7 91CDEGHGL 97 (SEQ ID NO: 20) Kp7-2 YCDEGHLCY (SEQ ID NO: 1) Kp7-3 YCDEGLCY(SEQ ID NO: 2) Kp7-4 YCDEGYFCY (SEQ ID NO: 3) Kp7-5 YCDEGEYCY (SEQ IDNO: 4) Kp7-6 YCDEHFCY (SEQ ID NO: 5) Kp7-7 YCDEHGLCY (SEQ ID NO: 6)Kp7-8 YCDEHGQCY (SEQ ID NO: 7) Kp7-9 YCDEKFCY (SEQ ID NO: 8) Kp7-10YCDEQFCY (SEQ ID NO: 9)

Example 2 Inhibition of FasL Binding to Fas Receptor

[0166] Fas mimetic activity was evaluated in an assay that measuredFasL-Flag binding (100 ng/ml) to Fas-Fc fusion protein immobilized ontoplastic plates. The first generation mimetics Kp1-1,2-2, 3-2,4-2 and 7-2were designed from different deduced binding sites of Fas to FasL andscreened using a binding inhibition assay comprising 300 μM of peptideand 20 nM of soluble Fas receptor (FIG. 2A). The results indicated thatKp7 loop is a preferred surface for the design of mimetics as atemplate. Additional generations of exocyclic peptides derived from theKp7 loop surface were also engineered. By the analysis of theinteraction site between FasL and Fas, and biological activity ofdifferent mimetics, the aspartic and glutamic acids in Kp7 loop appearto represent the most relevant residues involved in the interaction.However, the particular acidic amino acid at each position is notcritical. Hence, for example, either an aspartic acid or a glutamic acidresidue can be present at either position. Modification of otherresidues of Kp7 led to some improvement of inhibitory activities as seenwith the Kp7 series (FIG. 2A).

[0167] To compare the inhibitory activities of the Kp7 peptide series,dose-response studies were performed. The best activity was found withKp7-6, which inhibited 50% of the FasL-Flag molecule binding toimmobilized Fas-Fc at 150 μM (FIG. 2B).

Example 3 Binding Affinity and Specificity of Kp7 Mimetics

[0168] The kinetics of binding of Kp7-6, which mediated the bestinhibitory activity to FasL, was performed using surface plasmonresonance (BIAcore™) analysis. FasL-Flag was immobilized onto a sensorchip and various solutions containing different concentrations of Kp7-6were passed over the surface. FIG. 3A shows the sensogram resultobtained with these different Kp7-6 concentrations. The k_(on) andk_(off) rate constants were estimated to be 6.85×10 M⁻¹ s⁻¹ and7.65×10⁻⁴ s⁻¹, respectively, and a K_(D) of value of 1.12×10⁻⁵ M wasobtained from the ratio of the dissociation/association rate constants.The k_(off) value is considered as an important indicator in thedevelopment of therapeutics with biological activity (Benveniste, M. etal. Br J Pharmacol 104, 207-221 (1991); Yiallouros, I. et al. Biochem J331, 375-379 (1998)), and generally correlates with potent biologicaleffects. (Moosmayer, D. et al. J Interferon Cytokine Res 16, 471-477(1996)). Although the K_(D) of the Kp7-6-FasL interaction showed lessaffinity than that noted for Fas-FasL interactions (Starling, G. C. etal. J Exp Med 185, 1487-1492 (1997)), the k_(off) rate was similar withusual antigen-antibody interaction, which suggests that Kp7-6 forms astable receptor complex and may be useful practically. (Berezov, A. etal. J Med Chem 44, 2565-2574 (2001)).

[0169] To assess the specificity of Kp7-6 binding interaction, Fas, FasLand TNFα were immobilized on a sensor chip. Kp7-6 bound to FasL but notto TNFα, which indicates Kp7-6 bound to FasL specifically (FIG. 3B).Kp7-6 also bound to Fas (FIG. 3B). This observation is reminiscent offeatures of the soluble TNF receptor I (p55) which has been shown toform anti-parallel homodimeric complexes in the absence of ligand.(Naismith, J. H. et al. Structure 4, 1251-1262 (1996)). These resultssuggest that Fas may also form such antiparallel dimers and the Kp7 loopmay contribute to antiparallel dimer formation. No significant bindingto TNF-R was detected.

Example 4 Inhibition of FasL-Induced Cytoxicity

[0170] To evaluate the effect of mimetics on Fas-mediated cytotoxicity,FasL-sensitive Jurkat cells were stimulated with soluble FasL-Flagfusion protein in the presence or absence of various concentrations ofmimetics. The inhibitory effects of mimetics on Fas-mediatedcytotoxicity were consistent with the results of the FasL-bindinginhibition (FIG. 4). Kp7-6 showed a dose-dependent inhibitory activity.A concentration of 1 mM Kp7-6 protected more than 90% cells fromFas-mediated cytotoxicity (FIG. 4). Kp7-10 also showed a dose-dependentinhibition (FIG. 4). The cyclic peptides did not mediate anycytotoxicity for Jurkat cells in the range of concentration tested (datanot shown). Large dose of Kp7-6 was used in this experiment for bettersignal-to-noise ratio. To determine if mimetics derived from otherbinding sites would potentially be able to interact synergistically toblock the Kp7 surface, Kp7 was used in combination with mimetics.Neither Kp7-6 nor Kp7-10 used in combination with members of the Kp4mimetics showed any significant synergy, suggesting that inhibition ofthe Kp7 binding site by itself is sufficient to antagonize FasL activity(data not shown).

Example 5 Inhibition of FasL-Induced Apoptosis

[0171] Biological activity of Fas mimetics was evaluated further bydetermining the effect of mimetics on Fas-L induced apoptosis. Apoptosiswas measured by determining phosphatidylserine (PS) externalization onthe cell membrane using annexin V. Translocation of PS to the outerleaflet of the plasma membrane is a common feature of apoptosis and isan early event that can be quantitatively measured using annexin V-FITCbinding. Jurkat cells treated with FasL-Flag (200 ng/ml) for 3h showed amarked increase in PS exposure compared with untreated cells (FIG. 5).The increase in Jurkat cell apoptosis was prevented by Kp7-6 in adose-dependent manner (FIG. 5).

Example 6 Protection of Mice Against Con A-Induced Injury

[0172] The biological activity of mimetic Kp7-6 was measured in vivo.FasL induced apoptosis is one of the primary and dominant pathways bywhich liver cells undergo apoptosis under various conditions such asviral infection, drug toxicity and other lesions. (Kakinuma, C. et al.Toxicol Pathol 27, 412-420 (1999); Famularo, G., et al. Med Hypotheses53, 50-62 (1999); Gantner et al. supra). Several studies have found thatblocking Fas signaling either by RNA interference or byolgionucleotides, will limit the extent of liver damage. (Zhang, H. etal. Nat Biotechnol 18, 862-867 (2000); Song, E. et al. Nat Med (2003)).The antagonistic effect of Kp7-6 in vivo was tested using the ConA-induced hepatitis model. C57BL/6 mice were pretreatedintraperitoneally (IP) with anti-FasL monoclonal antibody, Fas mimeticpeptides or saline. After 30 min, animals were challenged intravenouslywith Con A or saline. The induction of liver damage and inflammatoryhepatitis was evaluated by measuring the serum activities of alanineaminotransferase (ALT) and aspartate aminotransferase (AST) 12 h afterCon A treatment. The activities of both transaminases were reduced inKp7-6 pretreated mice (FIGS. 6A-B), indicating Kp7-6 blockedFas-mediated hepatic injury in vivo. Kp1-1 did not block the hepaticinjury (FIGS. 6A-B). These results were consistent with in vitro data(shown in the preceding examples). Hence, a small rationally designedmolecule that can effectively disable Fas receptor functions in vivo.

[0173] Suppression of the disease in anti-FasL monoclonal antibodypretreated mice was incomplete, consistent with genetic studies usingFas-deficient lpr/lpr mice, which also showed incomplete suppression ofdisease in the same Con A-induce hepatitis model. (Tagawa et al. supra).Mechanisms other than that mediated by the Fas-FasL system may alsoinvolved in this process.

Example 7 Fas Mimetic Inhibits Angiogenesis in the Eye

[0174] Fas Kp7 mimetic was used topically to treat angiogenesis in amouse eye model. Angiogenesis was induced by placing pellets coveredwith fibroblast growth factor (FGF) that upon release in the eye lead toblood vessel growth. The degree of neovascularization was measured bythe diffusion of FITC-dextran from newly formed/neovascularized bloodvessels, using routine methods that are well known in the art. See, forexample, De Fouw et al., Microvasc. Res. 38, 136-147 (1989); Tiedeken &Rovainer, Microvasc. Res. 41, 376-389 (1991); Rizzo et al., Microvasc.Res. 49, 49-63 (1995). Topical administration of the Fas Kp7 mimetic (1mg/ml; 3 times/day for 7 days) led to virtual inhibition of new bloodvessel formation. Control treatment with a TNF inhibitor also showed noeffect.

Example 8 Fas Mimetic Agonist Activity in a Werner Syndrome Cell Line

[0175] Fas mimetic activity was further investigated in an assay thatmeasured apoptosis of cells treated with Fas mimetics of the invention.Fas mimetic activity was investigated in two particular cells lines:N6803, an immortalized EBV-transformed human B-lymphoblastoid cell line(Kataoka et al., Differentiation 62, 203-211 (1997)), and WS10201, anEBV-transformed B-lymphoblastoid cell line derived from a Wernersyndrome patient (see, Okada et al., Biol. Pharm. Bull. 21, 235-239(1998); Hanma et al., Mutat. Res. 520, 15-24 (2002)). Werner syndrome isa recessive genetic disorder that causes premature aging and an enhancedrisk of rare cancers (see, Goto et al., Hum. Genet. 105, 301-307(1999)).

[0176] Live cells were separated by lymphocyte separation medium incultured in wells of a 96-well plate (10⁵ cells per well) for sixteenhours in the presence of an agonistic anti-Fas IgM antibody. Suchantibodies are commercially available, e.g. from Beckman Coulter(Fullerton, Calif.). Cell were incubated either with anti-Fas IgMantibody alone or with the Fas mimetic Kp7-6 at doses of 10, 100 and1000 ng/ml. A control group of cells was also incubated in culturemedium alone (i.e., no anti-Fas IgM or Fas mimetic) under the sameconditions. Apoptosis was assessed by TUNEL method (Gavrieli et al., J.Cell Biol. 119, 493-501 (1992); Gorczyca et al., Cancer Res. 53,1945-1951 (1993); see, also, Ausubel et al. (Eds.) in Current Protocolsin Molecular Biology, at page 14.13.15).

[0177] FIGS. 7A-7B plots the apoptosis levels measured in N6803 andWS10201 cells, respectively. As expected, inclubation of the cell lineswith anti-Fas IgM increased apoptosis compared to cells incubated inmedium along. Incubation with both anti-Fas IgM and Fas mimetic did notsignificantly alter apoptosis in the N6803 cell line compared toincubation with anti-Fas IgM along (FIG. 7A). However, Fas mimeticexhibited a synergistic effect with anti-Fas IgM in the WS10201 cellline, greatly enhancing apoptosis compared to treatment with anti-FasIgM in the absence of Fas mimetic (FIG. 7B).

[0178] These data show that Fas mimetic of the invention can stimulateapoptosis in these circumstances, sugesting an agonistic effect incertain conditions. In particular, and without being limited by anyparticular theory or mechanism of action, receptors such as Fas arebelieved to exists in two states (see, Lef, Trends Pharmacol. Sci. 16,89-97 (1995)): an inactive monomer form and an active, signal competentdimer. At low concentrations, such as the concentrations used in theseexperiments, Fas mimetic is believed to bind to the signal competentdimer form of Fas but does not shift the receptor population to theinactive monomer state. In these conditions, particularly in the absenceof FasL, Fas mimetics of the invention are expected to exhibit at leasta transient stimulation of Fas activity and so have an agonistic effect.At higher concentrations, however, Fas mimetics are believed to shiftequilibrium of Fas to the signal incompetent monomer form, therebyinhibiting Fas signal and so having an antagonistic effect. Such“inverse agonist” properties are known in the art and have beendescribed for peptide mimetics of other receptors, such as the CD4receptor. See, for example, Horie et al., Exp. Mol. Pathol. 73, 93-103(2002).

Example 9 Experimental Protocols

[0179] Materials. Human recombinant TNFα was obtained from RocheDiagnostics (Indianapolis, Ind.). Flag-tagged soluble human Fas ligand(FasL-Flag) and human Fas extra cellular domain-IgGFc fusion protein(Fas-Fc) were purchased from Kamiya Biomedical (Seattle, Wash.). Humanrecombinant TNF-receptor (I) extracellular domain-IgGFc fusion protein(TNFRI-Fc) was obtained from R & D systems (Minneapolis, Minn.).Anti-Flag-HRP antibody, hydrogen peroxide solution,3,3′,5,5′-tetramethylbenzidine (TMBZ) and concanavalin A were from SigmaBiochemical Co. (St. Louis, Mo.).

[0180] Cell lines. American Type Culture Collection Jurkat cells weregrown in RPMI 1640 medium supplemented with 10% heat inactivated fetalcalf serum, L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin(100 μg/ml) at 37° C. in a humidified 5% CO2 atmosphere.

[0181] Mice. Eight-week-old C57BL/6 (B6) mice were purchased from CLEAJAPAN Inc. (Tokyo, Japan). All mice used were maintained under specificpathogen-free conditions in our animal facility.

[0182] Molecular Modeling. Computer modeling and the structural analysiswere performed using both QUANTA and INSIGHT (Molecular Simulation, SanDiego, Calif.). The model peptides designed were constructed from theirsequences and folding using CHARMM. The side chain of amino acidresidues were first positioned to permitted conformation using thePonders rotamer (Ponder, J. W. et al. J Mol Biol 193, 775-791 (1987))database provided in QUANTA. Then the folded peptides were minimized toconvergence with a dielectric constant set to 80. The molecular model ofthe human Fas/FasL complex was built by using Modeller 3.0 (Sali, A. etal. J Mol Biol 212, 403-428 (1990); Sali, A. et al. Trends in BiochemSci 15, 235-240 (1990)) using the crystal structure of TNF receptor andmolecular model of Fas (Bajorath supra) from Brookhaven database.(Bernstein, F. C. et al. J Mol Biol 112, 535-542 (1977)). Theconformation of loops were constructed using both loop search algorithmand CONGEN. (Bruccoleri, R. E. et al. Nature 335, 564-568 (1988)). Thequality of the model were assessed using Ramachandran plot (for phi, psiviolations) and profile analysis. (Zhang et al. supra). The Fas/FasLcomplex was optimized using rigid body minimization using XPLOR 3.1(Brünger, A. T. X-PLOR. Version 3.1. A System for X-ray Crystallographyand NMR. (Yale University Press, New Haven, Conn., 1992)) and INSIGHT.

[0183] Mimetics were designed from essential sequences of Fas-FasLinteractions. About five to seven amino acid sequences of Fas, have beenused to mimic the conformation of loops Kp1 to Kp7 as shown in Table 1.

[0184] Peptide synthesis and cyclization. Peptides were synthesized bysolid-phase methods, deprotected, and released from the resin usinganhydrous HF. Peptides were lyophilized and further purified by HPLCutilizing a C18 column and then relyophilized. Peptides were more than95% pure by HPLC analysis and mass spectrometry.

[0185] The peptides containing internal cysteine residues were refoldedand oxidized as described previously. (Takasaki et al. supra). Briefly,peptides were dissolved at 100 μg/ml in distilled water adjusted to pH8.0 by (NH₄)₂CO₃ and stirred at 4° C. until 95% formation ofintramolecular disulfide bonds had been confirmed by DTNB (SigmaBiochemical Co., St. Louis, Mo.). The cyclized peptides were lyophilizedand analyzed for purity by HPLC. These peptides showed greater than 90%purity by HPLC analysis.

[0186] Solid phase ligand binding assay. Fas-Fc fusion protein (250ng/ml) diluted in PBS was immobilized onto 96 well ELISA plate (Costar,High Wycombe, UK) by incubating overnight at 4° C. After blocking withPBS containing 1% skim milk overnight at 4° C. and subsequent washingwith PBS containing 0.05% Tween 20 (PBS-Tw), Flag-tagged soluble FasL(100 ng/ml)/peptide solution preincubated in PBS containing 1% skim milkfor 1 h at 37° C. was added onto the Fas-Fc coated wells. After 2 hincubation at room temperature, the plate was washed with PBS-Tw, andanti-FLAG(M2)-HRP antibody 1:2500 in PBS containing 1% skim milk wasadded. After 1 h incubation at room temperature, the plate was washedwith PBS-Tw, and the enzyme reaction was started by adding the substratesolution (0.1M sodium acetate buffer (pH 5.0) containing 100 μg/ml ofTMBZ and 0.005% (v/v) H₂O₂) and stopped with 2N H₂SO₄. The absorbance at450 nm was measured with an ELISA reader.

[0187] Biosensor analysis. All experiments were carried out on a BIAcore3000 instrument (Biacore A G, Uppsala, Sweden) at 25° C. using PBS, pH7.4, containing 0.005% surfactant P20 (Biacore A G) as the runningbuffer. FasL-Flag, Fas-Fc or TNFRI-Fc was immobilized on research-gradeCM5 sensor chips (Biacore AG) using standardN-ethyl-N-dimethylaminopropyl carbodiimid/N-hydroxysuccinimide coupling.Immobilization was performed in 10 mM sodium acetate buffer at pH 4.5for FasL-Flag and Fas-Fc, and at pH 4.0 for TNFRI-Fc. After coupling,excess N-hydroxysuccinimide groups were inactivated with ethanolamine.For binding studies, about 1500 resonance units (RU) of FasL-Flag,Fas-Fc and TNFRI-Fc were coupled to the chips. Surface plasmon resonance(SPR) measurements were carried out at a flow rate of 20 ml min⁻¹. Datawere analyzed with the BIA evaluation 3.0 software (Biacore AG). Thesensograms give values for the relative response in resonance units (RU)after background subtraction versus time in seconds. The associationphase injection time was 300 seconds followed by dissociation buffer.

[0188] Cytotoxicity assay. Twenty microliters of Jurkat cells at 1×10⁵cells/ml were plated in 96-well U-bottom plates. FasL-Flag (120 ng/ml inculture medium) was preincubated with equal volume of peptide sample inPBS for 1 h at 37° C., and 20 μl of mixture was added to each well.After an incubation period of 24 h, each culture was pulsed with 1 μCiof [³1H]-thymidine for 24 h before harvesting on glass fiber filters.Incorporation of the ³H-thymidine obtained with culture medium alone andwith 30 ng/ml of FasL was used as reference for 100% survival and 0%survival, respectively. Survival (%) by several doses of peptides wasplotted. Incorporation of the radioactive label was measured by liquidscintillation counting (Wallac, Finland) and expressed as the arithmeticmean counts per minute (cpm) of triplicate cultures.

[0189] Flow cytometry assay for apoptosis. Apoptotic cells were detectedby annexin V-FITC binding to phosphatidylserine (PS) expressed on thecell membrane in the early phase of apoptosis using a commercial kitpurchased from Roche (Indianapolis, Ind.). Briefly, 1×10⁵ Jurkat cellswere cultured with FasL-Flag (250 ng/ml) in the presence or absence ofpeptide sample for 3 h. The cells were then washed with PBS andresuspended in 100 μl of binding buffer containing optimal concentrationof calcium, FITC-conjugated annexin V and propidium iodide (PI) for 10min. at room temperature. After adding an additional 400 μl of bindingbuffer, cells were analyzed by FACScan (Becton Dickinson ImmunocytometrySystems, San Jose, Calif.). CELLQuest software programs (BectonDickinson Immunocytometry Systems, San Jose, Calif.) were used forcollecting and analyzing data. Early apoptotic cells were expressed aspercentage of cells positive for annexin V and negative for PI. 10,000cells were analyzed in each condition.

[0190] Administration of Con A and measurement of serum transaminaseactivity. Hepatic damage was induced by injection of a single dose of0.5 mg Con A dissolved in pyrogen-free saline and administered to micevia the tail vein. Anti-FasL monoclonal antibody (MFL-4; Kayagaki, N. etal. Proc Natl Acad Sci USA 94, 3914-3919 (1997)) or Fas mimetic peptide(Kp7-6 or Kp1-1) was diluted with pyrogen-free saline and injected in asingle dose intraperitoneally 30 minutes before Con A.

[0191] Blood samples were collected from mice at 12 h after Con Ainjection, and the serum was taken by centrifugation. Serum activitiesof alanine aminotransferase (ALT) and aspartate aminotransferase (AST)were measured by Lippi-Guidi's method (Iatrozyme TA-LQ: Dia-latron Inc.,Tokyo, Japan). (Lippi, U. et al. Clin Chim Acta 28, 431-437 (1970)).

[0192] Statistical analysis. Results are expressed as mean±SE, andanalyzed by the Student's t test or analysis of variance (ANOVA) whereappropriate. Post hoc comparisons were performed using Scheffe test. A95% confidence interval was used to define statistical significance.

REFERENCES CITED

[0193] Numerous references, including patents, patent applications andvarious publications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entirety andto the same extent as if each reference was individually incorporated byreference.

1 20 1 9 PRT Artificial Sequence Synthetic peptide derived from humanFas 1 Tyr Cys Asp Glu Gly His Leu Cys Tyr 1 5 2 8 PRT ArtificialSequence Synthetic peptide derived from human Fas 2 Tyr Cys Asp Glu GlyLeu Cys Tyr 1 5 3 9 PRT Artificial Sequence Synthetic peptide derivedfrom human Fas 3 Tyr Cys Asp Glu Gly Tyr Phe Cys Tyr 1 5 4 9 PRTArtificial Sequence Synthetic peptide derived from human Fas 4 Tyr CysAsp Glu Gly Glu Tyr Cys Tyr 1 5 5 8 PRT Artificial Sequence Syntheticpeptide derived from human Fas 5 Tyr Cys Asp Glu His Phe Cys Tyr 1 5 6 9PRT Artificial Sequence Synthetic peptide derived from human Fas 6 TyrCys Asp Glu His Gly Leu Cys Tyr 1 5 7 9 PRT Artificial SequenceSynthetic peptide derived from human Fas 7 Tyr Cys Asp Glu His Gly GlnCys Tyr 1 5 8 8 PRT Artificial Sequence Synthetic peptide derived fromhuman Fas 8 Tyr Cys Asp Glu Lys Phe Cys Tyr 1 5 9 8 PRT ArtificialSequence Synthetic peptide derived from human Fas 9 Tyr Cys Asp Glu GlnPhe Cys Tyr 1 5 10 8 PRT Artificial Sequence Synthetic peptide derivedfrom human Fas 10 Tyr Cys Asn Ser Thr Val Cys Tyr 1 5 11 10 PRTArtificial Sequence Synthetic peptide derived from human Fas 11 Tyr CysAsp Lys Ala Glu His Phe Cys Tyr 1 5 10 12 11 PRT Artificial SequenceSynthetic peptide derived from human Fas 12 Tyr Cys Asn Thr Arg Thr GlnAsn Thr Cys Tyr 1 5 10 13 9 PRT Artificial Sequence Synthetic peptidederived from human Fas 13 Tyr Cys Gln Glu Lys Glu Tyr Cys Tyr 1 5 14 10PRT Artificial Sequence Synthetic peptide derived from human Fas 14 TyrCys Gln Glu Arg Lys Glu Tyr Cys Tyr 1 5 10 15 319 PRT Homo sapiens 15Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser 1 5 1015 Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn 20 2530 Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro 35 4045 Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro 50 5560 Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 65 7075 80 Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly 8590 95 Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg100 105 110 Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His CysAsp 115 120 125 Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys ThrLeu Thr 130 135 140 Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser AsnLeu Gly Trp 145 150 155 160 Leu Cys Leu Leu Leu Leu Pro Ile Pro Leu IleVal Trp Val Lys Arg 165 170 175 Lys Glu Val Gln Lys Thr Cys Arg Lys HisArg Lys Glu Asn Gln Gly 180 185 190 Ser His Glu Ser Pro Thr Leu Asn ProGlu Thr Val Ala Ile Asn Leu 195 200 205 Ser Asp Val Asp Leu Ser Lys TyrIle Thr Thr Ile Ala Gly Val Met 210 215 220 Thr Leu Ser Gln Val Lys GlyPhe Val Arg Lys Asn Gly Val Asn Glu 225 230 235 240 Ala Lys Ile Asp GluIle Lys Asn Asp Asn Val Gln Asp Thr Ala Glu 245 250 255 Gln Lys Val GlnLeu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys 260 265 270 Glu Ala TyrAsp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys 275 280 285 Thr LeuAla Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser 290 295 300 AspSer Glu Asn Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu Val 305 310 315 16 6PRT Homo sapiens 16 Cys Asn Ser Thr Val Cys 1 5 17 9 PRT Homo sapiens 17Asp Lys Ala His Phe Ser Ser Lys Cys 1 5 18 5 PRT Homo sapiens 18 Cys ThrArg Thr Gln 1 5 19 7 PRT Homo sapiens 19 Cys Gln Glu Gly Lys Glu Tyr 1 520 7 PRT Homo sapiens 20 Cys Asp Glu Gly His Gly Leu 1 5

What is claimed is:
 1. A Fas mimetic which comprises an exocyclicpeptide having an amino acid sequence of a Fas surface domain thatinteracts with FasL.
 2. The mimetic according to claim 1 in which thesurface domain is a surface loop domain.
 3. The mimetic according toclaim 1 wherein the surface domain is selected from the group consistingof SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ IDNO:
 20. 4. A Fas mimetic represented by the formula (I)

wherein B₁ and B₉ are independently a peptide of 1-6 amino acids, atleast one of which is a hydrophobic amino acid, an aromatic moiety or aheteroaromatic moiety, Z₂ is a moiety that is capable of forming acovalent linkage with B₁, X₃ and Z₈, Z₈ is a moiety that is capable offorming a covalent linkage with B₉, X₇ and Z₂, X₃ is a hydrophilic aminoacid or a bond, X₄ is aspartic acid or glutamic acid, X₅ is asparticacid or glutamic acid, X₆ is selected from the group consisting ofhistidine, lysine, arginine, asparagine or glutamine, X₇ is an aromaticor heteroaromatic moiety, “—” is a covalent linkage comprising an amide,substituted amide or an isostere of amide thereof, and “═” is a covalentlinkage, or a pharmaceutically acceptable salt, metabolite or prodrugthereof.
 5. The mimetic of claim 4 wherein Z₂ and Z₈ are cysteineresidues.
 6. The mimetic of claim 5 wherein X₃ is a bond.
 7. The mimeticof claim 6 wherein X₄ is aspartic acid and X₅ is glutamic acid.
 8. Themimetic of claim 7 wherein X₇ is phenylalanine.
 9. The mimetic of claim8 wherein B₁ is —R₁-R₂, where R₁ is an aromatic amino acid linked to Z₁and R₂ is a peptide of 1-5 amino acids.
 10. The mimetic of claim 8wherein B₉ is —R₃-R₄, where R₃ is an aromatic amino acid linked to Z₈and R₄ is a peptide of 1-5 amino acids.
 11. The mimetic of claim 9wherein B₉ is —R₃-R₄, where R₃ is an aromatic amino acid linked to Z₈and R₄ is a peptide of 1-5 amino acids.
 12. The mimetic of claim 8wherein B₁ or B₉ is an aromatic amino acid.
 13. The mimetic of claim 12wherein each of B₁ and B₉ is an aromatic amino acid.
 14. The mimetic ofclaim 12 wherein B₁ or B₉ is tyrosine.
 15. The mimetic of claim 14wherein each of B₁ and B₉ is tyrosine.
 16. The mimetic of claim 4wherein said mimetic has a K_(D) for FasL of 10⁻⁴ M or less.
 17. Themimetic of claim 16 wherein said mimetic has a K_(D) for FasL of 10⁻⁵ Mor less.
 18. The mimetic of claim 4 wherein said mimetic has a k_(off)for FasL of 10⁻³ s⁻¹ or less.
 19. The mimetic of claim 4 wherein saidmimetic has a k_(off) for FasL of 10⁻⁴ s⁻¹ or less.
 20. The mimetic ofclaim 4 comprising a sequence selected from the group consisting ofYCDEGHLCY (SEQ ID NO: 1); YCDEGLCY (SEQ ID NO: 2); YCDEGYFCY (SEQ ID NO:3); YCDEGEYCY (SEQ ID NO: 4); YCDEHFCY (SEQ ID NO: 5); YCDEHGLCY (SEQ IDNO: 6); YCDEHGQCY (SEQ ID NO: 7); YCDEKFCY (SEQ ID NO: 8); and YCDEQFCY(SEQ ID NO: 9).
 21. The mimetic of claim 20 comprising a sequenceselected from the group consisting of YCDEHFCY (SEQ ID NO: 5); YCDEKFCY(SEQ ID NO: 8); and YCDEQFCY (SEQ ID NO: 9).
 22. The mimetic of claim 4consisting of sequence selected from the group consisting of YCDEGHLCY(SEQ ID NO: 1); YCDEGLCY (SEQ ID NO: 2); YCDEGYFCY (SEQ ID NO: 3);YCDEGEYCY (SEQ ID NO: 4); YCDEHFCY (SEQ ID NO: 5); YCDEHGLCY (SEQ ID NO:6); YCDEHGQCY (SEQ ID NO: 7); YCDEKFCY (SEQ ID NO: 8); and YCDEQFCY (SEQID NO: 9).
 23. The mimetic of claim 22 consisting of a sequence selectedfrom the group consisting of YCDEHFCY (SEQ ID NO: 5); YCDEKFCY (SEQ IDNO: 8); and YCDEQFCY (SEQ ID NO: 9).
 24. The mimetic of claim 23consisting of the sequence YCDEHFCY (SEQ ID NO: 5).
 25. A Fas mimeticcomprising a sequence selected from the group consisting of YCNSTVCY(SEQ ID NO: 10), YCDKAEHFCY (SEQ ID NO: 11), YCNTRTQNTCY (SEQ ID NO:12), YCQEKEYCY (SEQ ID NO: 13), and YCQERKEYCY (SEQ ID NO: 14).
 26. Apharmaceutical composition comprising a mimetic of any one of claims 1,4 or 25 and a pharmaceutically excipient.
 27. The pharmaceuticalcomposition of claim 26 wherein said excipient is a member selected fromthe group consisting of a diluent, buffer, carrier, stabilizer andpreservative.
 28. A method of treating a FAS-related pathologycomprising administering to a mammal suffering from said FAS-relatedpathology a therapeutically effective amount of the mimetic of any oneof claims 1, 4 or
 25. 29. The method of claim 28 wherein saidFAS-related pathology is an autoimmune pathology.
 30. The method ofclaim 28 wherein said FAS-related pathology is selected from the groupconsisting of rheumatoid arthritis, Sjogren's syndrome, multiplesclerosis, hepatitis, ocular disorder, renal injury, inflammation,aging, graft rejection and HIV infection.
 31. The method of claim 30wherein said FAS-related pathology is selected from the group consistingof hepatitis and ocular disorder.
 32. The method of claim 31 whereinsaid FAS-related pathology is macular degeneration.
 33. The method ofclaim 28 wherein said mammal is a human.
 34. The method of claim 28wherein said mimetic is administered as a pharmaceutically acceptablecomposition.
 35. The method of claim 33 wherein said mimetic isadministered via an oral, parenteral, intranasal, sublingual, rectal orinhalatory route, or by insufflation, transdermal patches or lyophilizedcomposition.
 36. The method of claim 35 wherein said mimetic isadministered in an amount of between about 0.01 to about 25 mg/kg/day.37. The method of claim 36 wherein said mimetic is administered in anamount of between about 0.1 to about 10 mg/kg/day.
 38. The method ofclaim 37 wherein said mimetic is administered in an amount of about 0.2to about 5 mg/kg/day.
 39. The method of claim 35 wherein said mimetic isadministered at a total daily dose of about 25 to about 1000 mg.
 40. Themethod of claim 39 wherein said mimetic is administered at a total dailydose of about 150 to about 500 mg.
 41. The method of claim 39 whereinsaid mimetic is administered at a total daily dose of about 350 mg. 42.A method of inhibiting Fas receptor-Fas ligand interaction comprisingexposing Fas or Fas L to an effective amount of a mimetic according toclaim 1, 4 or 25 to inhibit said interaction.
 43. The method of claim 41wherein the FAS receptor is present on the surface of a cell.
 44. Themethod of claim 42 wherein said exposure occurs in vitro.
 45. The methodof claim 42 wherein said exposure occurs in vivo.
 46. The method ofclaim 45 comprising administering said mimetic to a mammal comprising acell with FAS receptor on the cell surface.
 47. The method of claim 46wherein said mammal is a human.